The mechanisms that lead to tissue- and stage-specific patterns of gene expression are being investigated. Patterns of regulation of homologous alcohol dehydrogenase (ADH) genes in closely related species of picture-winged Drosophila are frequently dramatically different. These natural variants provide a model system for investigating general mechanisms of gene regulation. Genetic analysis has established that complex, cis-acting regulatory elements specify the alternative patterns of expression. The nature and function of these cis-acting regulators is being investigated at the molecular level using recombinant DNA methods. All tested variants affect gene expression at or near transcription. Transcripts are produced from two distinct initiation sites whose tissue and stage specific utilization also varies among the species studied. Differences in regulation are not related to gross chromosomal rearrangements, but significant differences are detected in comparative restriction maps. The structural comparisons will be extended by obtaining complete DNA sequences for the ADH gene and flanking regions from at least four species representing a range of regulatory patterns. Tissue- and stage-specific differences in chromatin structure (revealed by nuclease sensitivity) will be examined in the same four species and the location of specific features like hypersensitive sites will be related to the sequence comparisons. The distribution and production of alternative transcripts will be further characterized. Finally, methods for analyzing regulatory variants by interspecific P element mediated transformation will be explored. It is hoped that this will lead to functional tests of chimeric genes constructed in vitro and hence to a dissection of the elements specifying alternative patterns of regulation. The significance of this work for human health lies in the potential for better understanding of gene regulation and hence of conditions caused by failure of normal gene regulation, probably including many birth defects, metabolic disorders in which the level (rather than structure) of a particular enzyme is abnormal and some forms of cancer.